The broad goal of Project 3 is to devise and implement novel strategies of effective, low-toxicity EBV-specific T cell therapy for EBV-positive lymphomas, which account for approximately 40% of all human lymphomas. In a recent clinical trial of such immunotherapy in patients with high-risk active disease at the time of infusion of EBV-specific T cells (EBVSTs), we found that favorable tumor responses correlated with increased numbers of both EBVSTs and T cells that recognized nonviral tumor antigens (TAs), an example of antigen spreading. In most patients, however, the increases in both types of T cells were only transient, suggesting induction of T-cell anergy by potent immunosuppressive mechanisms in the tumor microenvironment. Thus, to prolong T cell expansion and function in this hostile setting, we are testing whether artificial costimulation by costimulatory chimeric antigen receptors (CoCARs) will enhance T cell proliferation and sustain EBVST activation in the face of inhibitory molecules. This approach, like that with classical CARs, combines the antigen binding domain of an antibody with costimulatory endodomains that trigger proliferation of the host T cell, but lacks the zeta chain of the TCR that is required to initiate cytotoxicity. The CoCAR therefore allows any cognate target cell to induce T cell costimulation without sustaining damage itself. CD19 was selected as the CoCAR target antigen because B cells are ubiquitous in lymphoid tissues and are often found within lymphoma sites; moreover, their function as professional antigen-presenting cells should enable them to enhance CoCAR signaling appreciably. The overarching hypothesis for this strategy ? that appropriate stimulation by EBV antigens and CD19 will render CoCAR-expressing EBVSTs resistant to tumor-derived inhibitory molecules, promoting their expansion and persistence after infusion and thus greater antigen spreading and better tumor responses ? will be tested in the following specific aims. AIM 1: Optimize in a preclinical model the CD19-specific CoCAR for use in human EBV-specific T cells . AIM 2: Evaluate the feasibility and safety of using EBVSTs modified with CD19-directed CoCARS to treat patients with EBV-associated Hodgkin lymphoma or non-Hodgkin lymphoma. AIM 3: Evaluate the expansion, persistence and antitumor activity of CoCAR-modified EBVSTs and TA- specific T cells, based on quantitative PCR measurements, ELIspot assay results, and imaging studies. Validation of this strategy may lead to its common use in the treatment of EBV-positive lymphoma.